Streptolysin S (SLS) is the non-antigenic, oxygen-insensitive cytolysin which is produced by Group A streptococci. It is largely responsible for a zone of beta-haemolysis surrounding colonies on blood agar media.   In this paper it is described, for the first time, reliable detection, visualization and quantification of SLS using blood agar overlay (zymogram) method. This technique is sensitive and protocol describes a method with broad potential to elucidate previously undetectable biological activity of streptomycin S (SLS) on gel electrophoresis.